| A new strategy of immunotherapy was developed to treat opioid -dependent mice. The morphine incorporated a succinic anhydride linker moiety in place of hydroxy group at C-6 of the morphine framework. Then morphine 6-hemisuccinate was conjugated to proteins in the presence of carbodi-imide (EDCI). The mice were immunized with vaccines in Freund's adjuvant. The characteristics of immunized mice and antiserum were evaluated comprehensively.1 Preparation of morphine vaccines: Free morphine base was prepared by morphine hydrochloride, and the yield was 83.2%. Reflux of morphine base with excess succinic anhydride in dry benzene led to the selective succinylation of the 6-hydroxy group, and then M-6-S (6-succinyl-morphine) was acquired. The yield was 64%. The Rf of morphine or M-6-S was 0.56 and 0.14 respectively by thin-layer chromatography (TLC). There was no significant different between M-6-S and morphine in UV spectrum. Four morphine vaccines showed good antigenicity.2 Immunological evaluations of morphine vaccines: Antibody titers were determined and the maintaining time of immune effects was monitored in immunized mice. The average titer of M-6-S-BSA group was obviously higher than those of other groups. Both of morphine and heroin could competitively inhibit the combination of M-6-S-BSA with antiserum from mice immunized with M-BC and M-6-S-BC, and the inhibition was in a dose-dependent manner. The cross-reactive assay suggested that the combination of antigen with antiserum could be inhibited by opioids, but not by other chemicals. It appeared the specificity of antiserum to opioids.3 Pharmacological evaluations of morphine vaccines: Developing a morphine-dependent model, the withdrawal symptoms were observed in mice immunized with four vaccines. Compared with the model group, jumping time was reduced significantly in M-6-S-KLH group (P<0.05), but there was5no difference (P>.05) in M-6-S-BSA group. Effects on withdrawal symptoms in mice immunized with M-BC and M-6-S-BC vaccines respectively were different significantly from the model group (P<0.05). Then the large dosages morphine-dependent model was developed, effects on withdrawal symptoms in M-BC and M-6-S-BC groups had no difference with the model group (P>0.05). It showed that the ability of neutralization of antiserum was limited in immunotherapy groups. In mice acetic acid writhing test, morphine decreased the number of writhing in 15min after administration of 0.6% acetic acid. But the mice immunized with M-BC and M-6-S-BC respectively administrated by morphine could not reduce the number of writhing compared with the morphine group (P<0.05). The serum or cerebral concentrations of morphine were assayed by radioimmunoassay, and the latter was reduced significantly (P<0.01). The weight of organs had no difference between immunized mice and the control mice. The addiction of morphine vaccines was lower than morphine (P<0.05). 4 Preparation of anti-morphine polyclonal antibodies and monoclonal antibodies: Antiserums of rabbits were collected after immunization, and the average liter was 1:64000. Antiserums were purified by affinity chromatography, and the titer was 16~80ng. Morphine could competitively inhibit the combination of purified antibodies with M-6-S-BSA. The purified antibodies of low and high dose could affect withdrawal symptom in the morphine-dependent mice and could reduced the cerebral concentrations of morphine. The monoclonal antibody of morphine is preparing by us.Conclusions: The research prepared morphine vaccine and developed an active immunization to prevent morphine from penetrating the blood-brain barrier, and it could offer a new means to block the action of opioids. The results suggested that immunotherapy might be a promising means to explore new treatments for opioid abuse. |
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